FIGS. 7 and 8 of the patent or application are photographs. Copies of this patent or patent application publication with photographs will be provided by the Office upon request and payment of the necessary fee.
The invention relates to a method for producing hemin proteins using plant cells, and in particular the hemin proteins capable of reversibly binding oxygen, for example hemoglobin and its derivatives, and myoglobin. It relates, in addition, to the proteins obtained using the method. The invention also relates to the genetically transformed cells and plants capable of producing these proteins, and to the nucleic acid constructs involved in the transformation. In addition, the invention relates to products, for example pharmaceutical or cosmetic products, containing these hemin proteins.
Hemin proteins are complex molecules composed of one or more polypeptide chain(s) in association with one or more ferroporphyrin nucleus or nuclei. These nuclei are composed of four pyrrole rings, juxtaposed in a closed structure and linked by methene bridges, and containing an iron atom at the center of the molecule. Hemin proteins differ from one another in the nature and the number of the polypeptide chains and in the nature of the side chains carried by the eight xcex2 atoms of the pyrrole rings. An example of a ferroporphyrin nucleus is iron-containing protoporphyrin IX, also known by the name xe2x80x9cprotohemexe2x80x9d or simply xe2x80x9chemexe2x80x9d (FIG. 1).
The hemin protein family comprises numerous substances which are important from the biological point of view in animals and in plants, particularly hemoglobin, myoglobin, cytochromes, peroxidases and catalases.
Hemoglobin is the main constituent of the red blood cells. Its essential function is to bind, transport and deliver the quantity of oxygen necessary for normal tissue function.
The hemoglobin molecule is composed of two types of globin chains or subunits, called xcex1 and xcex2 (of 141 and 146 amino acids respectively), and linked in pairs to form a xcex12xcex22 tetramer. Each of these subunits contains, solidly attached in a hydrophobic sac, a heme molecule (that is to say protoporphyrin IX) containing, at the center, a divalant iron atom (Fe2+) to which a molecule of oxygen reversibly binds. Each tetrameric hemoglobin molecule therefore contains 4 iron atoms and 4 oxygen molecules which it binds during its passage through the lungs. The molecular mass of the tetramer is 64,650 D. In man, the xcex1 and xcex2 chains are synthesized from two types of genes situated on chromosomes 16 and 11 respectively.
The term beta, or xe2x80x9cnonalphaxe2x80x9d, type chains covers not only the beta chains, but also the chains called epsilon, gamma or delta.
Normally, in adults, more than 95% of the hemoglobin consists of alpha2 beta2 tetramer, that is to say the association of two heterologous alpha-beta dimers, associated with the catalytic complex, heme. 2% to 3% of a hemoglobin consisting of alpha2 delta2 tetramers, and traces of fetal hemoglobin alpha2 gamma2 exist.
The tetrameric human hemoglobin molecule exists in two quaternary forms or structures depending on whether oxygen is bound or not to the iron atoms. In the presence of oxygen, hemoglobin is said to be in an R (for relaxed) state and its affinity for oxygen is high. In the absence of oxygen, hemoglobin is said to be in a T (for tense) state and its affinity for oxygen is 100 times lower (Perutz, 1970). The resultant affinity is linked to the equilibrium between the concentrations of R and T forms. The higher the concentration of hemoglobin in the T form at any level of oxygenation, the lower this affinity. The affinity of hemoglobin for oxygen is regulated by the cofactor-2,3-diphosphoglycerate (DPG), a small molecule derived from the metabolism of glucose and which binds to the xcex2 chains of tetrameric hemoglobin, reducing its affinity for oxygen.
The increase in the risks of infection by products derived from human blood (hepatitis, HIV) makes the development of an artificial oxygen carrier as substitute for blood transfusion necessary.
Techniques using recombinant DNAs have been proposed for producing the protein chains of globin.
The aim of the first techniques developed was essentially to cause the alpha and beta chains to be synthesized in E. coli separately (Naga{umlaut over (i )}and Thogen-Sen, 1987), involving cumbersome methods for separate expression of each of the chains. These methods could hardly be exploited on an industrial scale.
More recently, the expression of soluble and functional recombinant hemoglobin has been developed in E. coli (Hoffman et al., 1990, P.N.A.S., 87, 8521-8525) and Saccharomyces cerevisiae (Wagenbach et al., 1991, Biotechnology, 9, 57-61). Each of these systems has advantages and disadvantages. Indeed, the highest expression levels are obtained in E. coli which has, nevertheless, the disadvantage of producing endotoxins and of not cleaving the NH2 terminal methionines contrary to Saccharomyces cerevisiae. In the yeast, the yields of synthesis of hemoglobin are low (3 to 5%), compared with the 10-15% obtained in E. coli. This currently limits the use of yeast in the context of an industrial development plan.
The use of animal cells in culture or of transgenic animals as hosts for production has also been achieved (Swanson et al., Bio/Technology, May 1992, 10, page 55). It appears that these techniques cannot currently be exploited because of low expression levels and the risks of contaminations by viruses and by prions.
The technical problem which the present invention proposes to solve is to produce hemin proteins, and in particular hemoglobin and its derivatives, in a large quantity at low costs, without the risk of viral or subviral contaminations. The inventors have provided a solution to this problem by using plant cells as host for the transformation and the production.
Various teams have already taken an interest in the production of mammalian recombinant proteins in plant cells or in transgenic plants. For example, the specific expression, in rapeseed, of leuenkephalin has been obtained with expression levels of about 0.1% (Vanderkerckhove et al., Biotechnology, 1989, 7, 929-932).
In 1990, Sijmons et al., (Biotechnology, 1990, 8, 217-221) transferred the gene for human serum albumin into tobacco and potato cells. Regardless of the origin of the signal peptides (human or plant), human serum albumin levels of the order of 0.02% of the total proteins were obtained in the potato leaves, stems and tubers.
Other mammalian recombinant proteins have also been produced in plants: hepatitis B surface antigen (Mason et al., P.N.A.S., 1992, 89, 11745-11749); human interferon (Edelbaum J. of Interferon Res., 1992, 12, 449-453); a mouse antibody to Streptococcus mutans, an agent for dental caries (Hiatt and Mass., FEBS, 1992, 307, 71-75); an anti-Herpes antibody (Russel, 1994) and hirudin (Moloney, 1994).
All these research studies show that the production of mammalian recombinant proteins in plant cells is possible and that the mechanisms of protein synthesis from the DNA sequences are similar in animal cells and plant cells.
On the other hand, little information is available on the subject of the iron-containing porphyrins in plants, particularly on their structures, their synthesis pathways and the assembly of the porphyrin nuclei and the protein chains to form the hemin proteins. The production of recombinant molecules having the capacity to reversibly bind oxygen, and requiring the assembly, in the cell, of heterologous proteins and of endogenous plant porphyrins has never been described.
The invention relates to a method for producing recombinant hemin proteins using plant cells. According to the method of the invention, the plant cell is genetically modified so as to be able to express the protein component of a hemin protein. The porphyrin nucleus is produced by the cell endogenously, the assembling of the protein and porphyrin components taking place spontaneously by virtue of their high affinity for each other.
More particularly, the invention relates to a method for producing hemin proteins comprising the following steps:
i) introducing, into plant cells, one or more nucleic acid molecule(s) each of which comprises at least one sequence encoding a protein component of a hemin protein of animal origin or a variant or a portion of this protein component, and optionally a sequence encoding a selection agent;
ii) selecting the cells which have integrated the nucleic acid encoding the protein component;
iii) propagating the transformed cells, either in culture or by regenerating whole transgenic or chimeric plants;
iv) recovering, and optionally purifying, a hemin protein comprising a complex of the protein or proteins encoded by the abovementioned nucleic acid with at least one iron-containing porphyrin nucleus, or a plurality of these complexes.
The invention preferably relates to a method for producing hemin proteins comprising the following steps:
i) introducing, into plant cells, one or more nucleic acid molecule(s) each of which comprises at least one sequence encoding a protein component of a hemin protein of animal origin preferably capable of reversibly binding oxygen or a variant or a portion of this protein component, and optionally a sequence encoding a selection agent;
ii) selecting the cells which contain the nucleic acid encoding the protein component of the hemin protein;
iii) optionally, propagating the transformed cells, either in culture or by regenerating whole transgenic or chimeric plants;
iv) recovering, and optionally purifying, a hemin protein comprising a complex consisting of the protein or proteins encoded by the abovementioned nucleic acid and at least one iron-containing porphyrin nucleus, or a plurality of these complexes.
In the context of the present invention, the term xe2x80x9chemin proteinxe2x80x9d means any protein having an iron-containing porphyrin nucleus as prosthetic group, and in particular protoporphyrin IX as exists in human myoglobin and hemoglobin (FIG. 1). The porphyrin nucleus may also be derivatives of heme from those of human heme. The side chains are preferably hydrophobic.
The hemin proteins of the invention include in particular the hemin proteins having, as main function, the reversible binding of oxygen, that is to say myoglobin and hemoglobin, as well as the cytochromes whose role is to transport electrons. The derivatives of these proteins conserving these functions are also included in the invention.
According to a preferred variant, the hemin protein of the invention is hemoglobin or a hemoglobin-type protein. In the context of the invention, the term xe2x80x9chemoglobin-type proteinxe2x80x9d includes all the hemin proteins having at the same time:
i) one or more xcex1- and/or xcex2-globin chain(s) or variants of these polypeptides, and
ii) one or more molecules of iron-containing protoporphyrin IX, or of protoporphyrins differing from protoporphyrin IX in the nature of the side chains,
iii) having a capacity to reversibly bind oxygen, preferably with an affinity of between 10 and 50 mm Hg at 37xc2x0 C., pH7.4. More particularly, the affinity is between 20 and 30 mm Hg, by way of example, the P50 of total blood at pH 7.2 is of 26xc2x12 mm Hg.
In the text which follows, the term xe2x80x9chemoglobin-type moleculexe2x80x9d will be used synonymously with the term xe2x80x9chemoglobin derivativexe2x80x9d.
In this context, a xe2x80x9cvariantxe2x80x9d of a protein component, and particularly of xcex1- or xcex2-globin, means an amino acid sequence which distinguishes itself in relation to the natural sequence by one or more amino acid substitution(s), deletion(s) or insertion(s). In general, the variant exhibits at least 90%, and preferably at least 95%, homology or identity with the natural sequence. In the context of the present invention, the percentage homology between two amino acid sequences is calculated as being the number of identical amino acids plus the number of similar amino acids in the alignment of the two sequences, divided by the length of the sequences between two given positions. If, between the two given positions, the two sequences do not have the same length, the percentage homology is the number of identical and similar amino acids, divided by the length of the longest sequence. The amino acids considered to be xe2x80x9csimilarxe2x80x9d are known in the art, see for example R. F. Feng, M. S. Jobson and R. F. Doolittle; J. Mol. Evol.; 1985; 21; 112-115. They are normally considered to be those which, within a permutation matrix, have a positive coefficient of substitution.
The term xe2x80x9cvariantxe2x80x9d also includes fragments of polypeptide chains, for example of xcex1- or xcex2-globin, normally having a length of at least 90% of the parent molecule. The variants can also be made longer than the parent molecule by adding nonfunctional sequences. Preferably, the variants conserve the biological and immunological properties of the parent molecule.
The first stage of the method of the invention consists in introducing, into plant cells, one or more nucleic acid molecule(s) comprising at least one sequence encoding a protein component of a mammalian hemin protein, or a variant of this component.
When the hemin protein is a single-chain protein, for example myoglobin or cytochrome, the nucleic acid introduced into the plant cells normally comprises a copy of the sequence encoding this protein.
On the other hand, when it is an oligomeric or a multimeric protein, such as hemoglobin or hemoglobin-type molecules, the sequences encoding the various protein units are introduced into the plant cell, either within the same nucleic acid molecule, or within separate nucleic acid molecules. Preferably, for the production of hemoglobin and its derivatives, the sequences encoding xcex1- and xcex2-globin, or their variants, are within the same vector, called co-expression vector. The vector may comprise one or more copy(ies) of each coding sequence.
Alternatively, the sequences encoding xcex1- and xcex2-globin, or their variants, may be present on separate nucleic acid molecules. According to this variant, the two molecules may be introduced into the same plant cell, provided that an appropriate selection system is available. Another technique consists in introducing one of the molecules into a first plant cell, and the other into a second plant cell. Each of the transformed cells is then regenerated into a whole plant, it then being possible for the plants thus obtained to be crossed in order to give a progeny capable of producing both the xcex1 and xcex2 chains. This approach can be used to optimize the yield of hemoglobin.
The nucleic acid molecules introduced into the plant cell during the first stage of the method are also part of the invention. Generally, these nucleic acids comprise:
i) one or more sequence(s) encoding a protein component of an animal hemin protein, and
ii) one or more sequence(s) encoding a targeting signal of plant origin, and/or sequences for regulation of transcription which are recognized by a plant cell.
More particularly, the nucleic acid of the invention comprises:
i) one or more sequence(s) encoding a protein component of an animal hemin protein, the said protein having the capacity to reversibly bind oxygen, and
ii) sequences for regulation of transcription which are recognized by a plant cell, comprising a promoter and sequences for regulation of termination, and
iii) one or more sequence(s) encoding a targeting signal of plant origin.
Preferably, the sequences encoding the protein component encode animal xcex1- or xcex2-globin, for example of human or bovine origin, or the variants thereof. In this manner, the properties of the molecule, and in particular the affinity for oxygen and the stability, can be optimized.
Among these modifications, it is possible, for example, to introduce into one or into both of the xcex1- and xcex2-globin chains, by site-directed mutagenesis, one or two sequence difference(s) in order to reduce the affinity for oxygen. These mutations may be chosen from examples of natural mutations (see Table I), or from the mutations indicated by examination of the three-dimensional model of natural hemoglobin A.
In a very advantageous manner, the mutants whose functional properties correspond to the physiological conditions for oxygen transport will be used: reversible binding, cooperativity and low speed of autooxidation. Among the mutants, there will be preferably used the double mutants xcex12xcex22F41Y,K82D (that is to say a mutant whose xcex2 chain comprises the following modifications: Phe-41 is replaced by Tyr, and Lys-82 is replaced by Asp) or xcex12xcex22F41Y,K66T (that is to say a mutant whose xcex2 chain comprises the following modifications: Phe-41 is replaced by Tyr, and Lys-66 is replaced by Thr) which correspond to these functional characteristics.
The modification of the xcex1 and xcex2 chains may also be carried out in order to stabilize the molecule, that is to say to avoid the dissociation of the tetramer into small-sized dimers which are rapidly filtered by the kidneys and which limit the intravascular life of hemoglobin. Covalent bridging, with the aid of phosphate or diaspirin, has been demonstrated as being an effective technique for stabilizing the tetramer (Benesch and Kwong, 1994). The same result can be obtained through modifications of the amino acid chain. The xcex1 subunits are produced in an alphaxe2x80x94alpha dimeric form linked by a glycyl residue. In this form, they conserve their capacity to correctly assemble onto the beta partner subunits and onto heme in order to form a soluble hemoglobin. This hemoglobin can no longer dissociate into dimers because the tetrameric structure is stabilized by a covalent bond (peptide bond) between the alpha-beta dimers. This technique makes it possible to increase the intravascular half-life of the molecule.
Among the variants, it is also possible to use a hybrid protein composed of a portion of the alpha chain and a portion of the beta chain.
According to a preferred variant of the invention, the nucleic acid comprises, in addition to the sequences encoding xcex1- or xcex2-globin, sequences encoding targeting signals. Preferably, these signals are chloroplast or mitochondrial targeting signals. The expression and/or accumulation of the recombinant proteins in these organelles is particularly preferred because of the availability of endogenous iron-containing porphyrins which are found here. The yield of hemin proteins is therefore increased. In addition, the targeting of the proteins toward the chloroplasts and the mitochondria avoids glycosylation of the protein, which may be advantageous since the natural hemoglobin molecule is not glycosylated.
As an example of chloroplast targeting signals, there may be mentioned the sequence encoding the transit peptide of the precursor of the small subunit of ribulose 1,5-diphosphate carboxylase of Pisum sativum (see examples). As mitochondrial targeting signals, there may be mentioned the sequence encoding the transit peptide of the precursor of the beta subunit of mitochondrial ATP-aseF1 of Nicotiana plumbaginifolia (see examples).
These transit peptides, as well as the N-terminal methionine, are normally cleaved in the chloroplasts or the mitochondria. The expression of the proteins in the plastids therefore also has the advantage of producing a molecule lacking N-terminal methionine as natural molecule.
According to another variant, the targeting sequences may be sequences encoding an N-terminal signal peptide (xe2x80x9cprepeptidexe2x80x9d), optionally in association with a signal responsible for retaining the protein in the endoplasmic reticulum (KDEL-type signal), or a vacuolar targeting signal or xe2x80x9cpropeptidexe2x80x9d. The presence of the N-terminal signal peptide or prepeptide allows the penetration of the nascent protein into the endoplasmic reticulum where a certain amount of post-translational processing occurs, particularly the cleaving of the signal peptide, the N-glycosylations, if the protein in question has N-glycosylation sites, and the formation of disulfide bridges. Among these various signals, the prepeptide responsible for the targeting of the protein into the endoplasmic reticulum, is dominant. It is normally a hydrophobic N-terminal signal peptide having between 10 and 40 amino acids and being of animal or plant origin. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, plant extensin, xcex1-mating factor, pathogenesis protein 1 or 2.
Normally, the signal peptide is cleaved by a peptidase signal upon the co-translational introduction of the nascent polypeptide into the lumen of the RER. The mature protein no longer contains this N-terminal extension.
The targeting sequences can, besides the prepeptide, also comprise an endoplasmic retention signal, consisting of the KDEL, SEKDEL or HEKDEL peptides. These signals normally exist at the C-terminal end of the protein and remain on the mature protein. The presence of this signal tends to increase the recombinant protein yields.
The targeting signals may, besides the prepeptide, also comprise a vacuolar targeting signal or xe2x80x9cpropeptidexe2x80x9d. In the presence of such a signal, after passing into the RER, the protein is targeted toward the vacuoles of the aqueous tissues, the leaves for example, as well as to the protein bodies of the storage tissues, for example the seeds, tubers and roots. The targeting of the protein toward the protein bodies of the seed is particularly advantageous because of the capacity of the seed to accumulate proteins, up to 40% of the proteins relative to the dry matter, in cellular organelles derived from the vacuoles, called protein bodies and because of the possibility of stocking, for several years, the seeds containing the recombinant proteins in the dehydrated state.
As propeptide, it is possible to use a signal of animal or plant origin, the plant signals being particularly preferred, for example prosporamine. The propeptide may be N-terminal (xe2x80x9cN-terminal targeting peptidexe2x80x9d or NTTP), or C terminal (CTTP) Since the propeptides are normally cleaved upon entry of the protein into the vacuole, it is not present in the mature protein.
The use of the signal peptide or prepeptide can lead to the glycosylation of the protein. Normally, globin has no N-glycosylation sites, but these may be introduced by mutagenesis. The xcex1 and xcex2 chains can also have O-glycosylation sites.
In the absence of any targeting signal, the protein is expressed in the cytoplasm.
The nucleic acid introduced into the plant cell may also comprise sequences for regulation of transcription which are recognized by the plant cell. The nucleic acid is in this case a xe2x80x9cchimeric genexe2x80x9d. The regulatory sequences comprise one or more promoter(s) of plant or viral origin or obtained from Agrobacterium tumefaciens. They may be constitutive promoters, for example the CaMV 35S, the double 35S, the Nos or OCS promoters, or promoters specific for certain tissues such as the grain or specific for certain phases of development of the plant. As promoters specific for seeds, there may be mentioned the promoter of the gene for napin and for the acyl carrier protein (ACP) (EP-A-0,255,378), as well as the promoters of the AT2S genes of Arabidopsis thaliana, that is to say the PAT2S1, PAT2S2, PAT2S3 and PAT2S4 promoters (Krebbers et al., Plant Physiol., 1988, vol. 87, pages 859-866). It is particularly preferable to use the cruciferin or phaseolin promoter or pGEA1 and pGEA6 of Arabidopsis, promoters of genes of the xe2x80x9cEm, Early Methionine labelled proteinxe2x80x9d type, which is strongly expressed during the phases of drying of the seed.
It is possible to envisage using xe2x80x9cenhancersxe2x80x9d to improve the efficiency of expression. When the transformation occurs directly in the chloroplast and mitochondrial genomes, gene promoters specific for these compartments can be used.
The sequences for regulation of transcription normally comprise sequences for termination of transcription which are of plant or of viral origin, for example 35S, or of bacterial origin
(Agrobacterium).
When the transforming nucleic acid does not comprise regulatory sequences, it is preferable to add onto each end of the nucleic acid a DNA sequence homologous to the genomic sequences which are adjacent to a specific insertion site in the genome. This allows the integration of the construct by homologous recombination, at a site where endogenous regulatory sequences can control the expression of the heterologous sequences.
The nucleic acids of the invention may also comprise one or more intron(s), preferably of plant origin. These introns, which are obtained from a plant gene, are introduced artificially so as to increase the efficiency of expression of the heterologous sequence.
Indeed, it has been demonstrated, particularly in monocotyledonous plants, that the insertion of an intron into the untranslated 5xe2x80x2 portion of a gene, that is to say between the site of initiation of transcription and the site of initiation of translation, leads to an improvement in the stability of the messenger, and consequently, to a better expression. The intron(s) used in this manner are obtained preferably from a monocotyledonous plant such as maize. This is preferably, but not necessarily, the first intron of the gene.
The nucleic acid sequence encoding xcex1- and xcex2- globin (SEQ ID NO: 30 and SEQ ID NO: 32, respectively) and its variants is normally cDNA. Appropriate sequences are illustrated in FIGS. 2 and 3, any degenerate sequence can also be used as well as the sequences of the variants as defined above.
The introduction of a nucleic acid molecule(s) into the plant cell can be carried out in a stable manner either by transformation of the nuclear genome, or by transformation of the chloroplast genome of the plant cell, or by transformation of the mitochondrial genome.
For the transformation of the nuclear genome, conventional techniques may be used. All known means for introducing foreign DNA into plant cells may be used, for example Agrobacterium, electroporation, protoplast fusion, particle gun bombardment, or penetration of DNA into cells such as pollen, microspore, seed and immature embryo. Viral vectors such as the Gemini viruses or the satellite viruses may also be used as introducing means. Agrobacterium tumefaciens and rhizogenes constitute the preferred means. In this case, the sequence of the invention is introduced into an appropriate vector with all the necessary regulatory sequences such as promoters, terminators and the like, as well as any sequence necessary for selecting the transformants which have integrated the heterologous sequences.
The transformation of the nuclear genome of the plant cell is often carried out using the targeting signals mentioned above and which determine the cellular compartment where the expression and/or accumulation of the protein will occur.
According to another variant of the invention, the introduction of the nucleic acid into the plant cell can be carried out by the transformation of the mitochondrial or chloroplast genomes (see for example Carrer et al., Mol. Gen. Genet., 1993, 241, 49-56).
Techniques for direct transformation of the chloroplasts or the mitochondria are known per se and may comprise the following steps:
i) introducing transformant DNA by the biolistic technique (Svab et al., P.N.A.S., 1990, 87, 8526-8530);
ii) integrating the transformant DNA by two homologous recombination events;
iii) selectively removing copies of the wild-type genome during repeated cell divisions on selective medium.
In order to allow the homologous recombination of the transformant DNA, two DNA fragments homologous to the genomic sequences, for example the rbcL and ORF 512 genes are added to each end of the DNA to be inserted into the genome.
The direct transformation of the chloroplasts or mitochondria has the advantage of substantially increasing the yield of hemoglobin but the N-terminal methionine is retained.
According to another variant of the invention, the heterologous nucleic acid can be introduced into the plant cell by means of a viral vector.
The method of the invention comprises a step of detecting the hemin proteins and in particular hemoglobin and its derivatives. This makes it possible to verify if the plant or the plant cell is capable, not only of expressing the heterologous proteins, but also of assembling them correctly with the porphyrin nucleus. For the hemoglobin in a complex environment containing other chromophores or molecules which scatter light, detection by time-resolved optical spectroscopy will be advantageously used. This technique is described in detail in the examples. Other detection techniques consist in using antibodies specific for the alpha or beta globin chains or their variants. The spectrometric and immunological techniques can be used in association with each other. The use of these techniques makes it possible to select the plants which are capable of producing hemoglobin and its derivatives according to the invention.
The method of the invention comprises, in addition, a step of recovering or extracting hemoglobin or its derivatives from plant tissues. The extraction is normally carried out by grinding the tissues, for example leaves or grains, in an appropriate buffer, filtering the ground product, precipitating the proteins in the supernatant, centrifuging and taking up the pellet in an appropriate buffer with dialysis. A partial purification step can also be carried out at this stage by chromatography on an anion-exchange column.
The tetramer of hemoglobin, or of its derivatives, is purified by two successive chromatographies on an ion-exchange resin followed by a step of concentrating and saturating the concentrate with carbon monoxide. These techniques are described in detail in the examples.
When the expression of hemoglobin and of its derivatives takes place under the control of a constitutive promoter, such as the 35S double promoter, an expression level of at least 1% hemoglobin compared with the total proteins may be obtained. The proteins represent about 10% of the dry mass of the leaf and a ton of dry tobacco leaves is harvested per hectare. It is therefore possible to obtain of the order of 100 grams of hemoglobin per hectare of tobacco cultivated, assuming that only 10% of the hemoglobin produced is purified.
The method of the invention therefore allows the production of hemoglobin at very low costs with a higher production capacity than that obtained using fermenters of the culture of bacteria or yeast.
Besides the method of transformation, the invention also includes vectors comprising one or more nucleic acid(s) or chimeric gene(s) defined above. As an example of vectors, there may be mentioned binary vectors or plasmids, viral vectors such as gemini viruses or the CaMVs.
The invention also relates to the plant cells transformed with the nucleic acid sequences of the invention. Preferably, they are transformed plant cells capable of producing one or more hemoglobin(s) or derivatives of hemoglobin according to the invention.
They may be plant cell cultures in vitro, for example in liquid medium. Various modes of culture (xe2x80x9cbatchxe2x80x9d, xe2x80x9cfed batchxe2x80x9d or continuous) for this type of cells are currently under study. The xe2x80x9cbatchxe2x80x9d cultures are comparable to those carried out in an Erlenmeyer flask since the medium is not changed, these cells thus have only a limited quantity of nutrient materials. The xe2x80x9cfed batchxe2x80x9d culture corresponds, for its part, to a xe2x80x9cbatchxe2x80x9d culture with programmed supply of substrate. For a continuous culture, the cells are supplied continuously with nutrient medium. An equal volume of the biomass-medium mixture is removed in order to maintain the volume of the reactor constant. The quantities of plant biomass which can be envisaged with cultures in bioreactors are variable depending on the plant species, the mode of culture and the type of bioreactor. Under certain conditions, biomass densities of about 10 to 30 g of dry weight per liter of culture can be obtained for species such as Nicotiana tabacum, Vinca rosea and Catharanthus roseus. 
The cells of the invention can also be immobilized, which makes it possible to obtain a constant and prolonged production of hemoglobin. The separation of the hemoglobin and the plant biomass is also facilitated. As immobilization method, there may be mentioned immobilization in alginate or agar beads, inside polyurethane foam, or alternatively inside hollow fibers.
The cells of the invention may also be root cultures. The roots cultivated in vitro, in a liquid medium, are called xe2x80x9cHairy rootsxe2x80x9d, they are roots transformed by the bacterium Agrobacterium rhizogenes. 
Instead of producing the hemoglobin of the invention by culturing plant cells, it is possible to regenerate chimeric or transgenic plants from transformed explants, using techniques known per se.
As appropriate plants, there may be mentioned the Angiospermae comprising monocotyledonous and dicotyledonous plants. More particularly, there may be mentioned tobacco, species belonging to botanic families such as leguminous plants (for example beans, peas and the like), cruciferous plants (for example cabbage, raddish, rapeseed and the like), Solanaceae (for example tomatoes, potato and the like), Cucurbitaceae (for example melon), Chenopodiaceae (for example beetroot), Umbelliferae (for example carrots, celery and the like). There may also be mentioned cereals such as wheat, maize, barley, triticale and rice, oleaginous plants such as sunflower and soybean. Tobacco, potato, tomato and maize are particularly preferred. For potato, the expression takes place preferably in the tubers.
The invention also relates to the seeds of transgenic plants capable of producing hemoglobin as well as their progeny.
The invention also relates to the hemin proteins which may be obtained by the method of the invention, in particular the hemin proteins capable of reversibly binding oxygen, for example the hemoglobins and derivatives thereof.
The hemoglobins of the invention are capable of binding O2 in a reversible manner with an affinity (P50) preferably close to physiological values (37xc2x0 C.), pH 7.40). The affinity of the molecule for O2 is expressed as P50: that is to say the partial pressure of O2 when hemoglobin or its derivatives is 50% saturated. The P50 is measured according to the usual techniques, for example by means of an analyzer which measures the percentage O2 saturation as a function of the O2 pressure (Kister et al., 1987). Normally, the hemoglobins of the invention have an acceptable autooxidation rate in order to minimize the formation of methemoglobin which is unsuited to the transport of O2. This characteristic can be measured by the absorption spectrum.
Preferably, the hemoglobins of the invention are tetramers, preferably alpha2 beta2, beta4, or optionally tetramers of chimeric xcex1/xcex2 subunits (Dumoulin et al., 1994, Art. Cells, Blood Subst., and Immob. Biotech., 22, 733-738) or multiples of four subunits. The physical size of the complex should be at least that of the tetramer in order to avoid its filtration by the kidneys.
The hemin proteins of the invention can be used in numerous pharmaceutical, cosmetic or industrial applications. The invention relates in particular to pharmaceutical compositions comprising one or more hemin protein(s) according to any one of claims 15 to 23, in association with a physiologically acceptable excipient.
In the pharmaceutical field, all the conditions requiring an improvement in the transport of oxygen can be treated with the hemoglobins of the invention, these conditions comprising the following:
acute or chronic hemorrhage,
states of shock,
coronary or sylvian angioplasties,
treatments of solid tumors, sensitization to gamma-therapy,
preservation of organs before transplant and during transport,
malignant hemopathies.
The hemoglobins of the invention are normally used in the form of an injection in solutions optionally stabilized as regards the tetrameric form of the complex (for example addition of pyridoxal phosphate or diaspirin) as regards autooxidation. It is also possible to use suspensions of hemoglobin grafted on a support in order to increase the lifetime in the bloodstream. The support may be any conventional support in this domain, for example polysaccharides.